The invention relates to methods, devices, and kits for obtaining and assaying biological samples from mammary fluid. More specifically, the invention relates to methods, devices, and kits for obtaining and assaying fluid and cytological samples from the mammary glands of a mammalian subject for evaluating, diagnosing and managing breast disease, including infections, pre-cancerous conditions, cancer susceptibility and cancer.
Breast cancer is by far the most common form of cancer in women, and is the second leading cause of cancer death in humans. Despite many recent advances in diagnosing and treating breast cancer, the prevalence of this disease has been steadily rising at a rate of about 1% per year since 1940. Today, the likelihood that a women living in North America will develop breast cancer during her lifetime is one in eight.
The current widespread use of mammography has resulted in improved detection of breast cancer. Nonetheless, the death rate due to breast cancer has remained unchanged at about 27 deaths per 100,000 women. All too often, breast cancer is discovered at a stage that is too far advanced, when therapeutic options and survival rates are severely limited. Accordingly, more sensitive and reliable methods are needed to detect small (less than 2 cm diameter), early stage, in situ carcinomas of the breast. Such methods should significantly improve breast cancer survival, as suggested by the successful employment of Papinicolou smears for early detection and treatment of cervical cancer.
In addition to the problem of early detection, there remain serious problems in distinguishing between malignant and benign breast disease, in staging known breast cancers, and in differentiating between different types of breast cancers (e.g. estrogen dependent versus non-estrogen dependent tumors). Recent efforts to develop improved methods for breast cancer detection, staging and classification have focused on a promising array of so-called cancer xe2x80x9cmarkers.xe2x80x9d Cancer markers are typically proteins that are uniquely expressed (e.g. as a cell surface or secreted protein) by cancerous cells, or are expressed at measurably increased or decreased levels by cancerous cells compared to normal cells. Other cancer markers can include specific DNA or RNA sequences marking deleterious genetic changes or alterations in the patterns or levels of gene expression associated with particular forms of cancer.
A large number and variety of breast cancer markers have been identified to date, and many of these have been shown to have important value for determining prognostic and/or treatment-related variables. Prognostic variables are those variables that serve to predict disease outcome, such as the likelihood or timing of relapse or survival. Treatment-related variables predict the likelihood of success or failure of a given therapeutic plan. Certain breast cancer markers clearly serve both functions. For example, estrogen receptor levels are predictive of relapse and survival for breast cancer patients, independent of treatment, and are also predictive of responsiveness to endocrine therapy. Pertschuk et al., Cancer 66:1663-1670, 1990; Parl and Posey, Hum. Pathol. 19:960-966, 1988; Kinsel et al., Cancer Res. 49:1052-1056, 1989; Anderson and Poulson Cancer 65:1901-1908, 1989.
The utility of specific breast cancer markers for screening and diagnosis, staging and classification, monitoring and/or therapy purposes depends on the nature and activity of the marker in question. For general reviews of breast cancer markers, see Porter-Jordan et al., Hematol. Oncol. Clin. North Amer. 8:73-100, 1994; and Greiner, Pharmaceutical Tech., May, 1993, pp. 28-44. As reflected in these reviews, a primary focus for developing breast cancer markers has centered on the overlapping areas of tumorigenesis, tumor growth and cancer invasion. Tumorigenesis and tumor growth can be assessed using a variety of cell proliferation markers (for example Ki67, cyclin D1 and proliferating cell nuclear antigen (PCNA)), some of which may be important oncogenes as well. Tumor growth can also be evaluated using a variety of growth factor and hormone markers (for example estrogen, epidermal growth factor (EGF), erbB-2, transforming growth factor (TGF), which may be overexpressed, underexpressed or exhibit altered activity in cancer cells. By the same token, receptors of autocrine or exocrine growth factors and hormones (for example insulin growth factor (IGF) receptors, and EGF receptor) may also exhibit changes in expression or activity associated with tumor growth. Lastly, tumor growth is supported by angiogenesis involving the elaboration and growth of new blood vessels and the concomitant expression of angiogenic factors that can serve as markers for tumorigenesis and tumor growth.
In addition to tumorigenic, proliferation and growth markers, a number of markers have been identified that can serve as indicators of invasiveness and/or metastatic potential in a population of cancer cells. These markers generally reflect altered interactions between cancer cells and their surrounding microenvironment. For example, when cancer cells invade or metastasize, detectable changes may occur in the expression or activity of cell adhesion or motility factors, examples of which include the cancer markers Cathepsin D, plasminogen activators, collagenases and other factors. In addition, decreased expression or overexpression of several putative tumor xe2x80x9csuppressorxe2x80x9d genes (for example nm23, p53 and rb) has been directly associated with increased metastatic potential or deregulation of growth predictive of poor disease outcome.
Additional representative breast disease markers within these various classes include prostaglandin E2 (PGE2); estrogen-regulated proteins such as pS2; interleukins (e.g., IL-10); S-100 protein; vimentin; epithelial membrane antigen; prostate specific antigen (PSA); bcl-2; CA15-3 (an aberrant form of polymorphic epithelial mucin (PEM)); CA 19-9; mucin core carbohydrates (e.g., Tn antigen and Tn-like antigens); alpha-lactalbumin; lipidassociated sialic acid (LASA); galactose-N-acetylgalactosamine (Gal-GaINAC); GCDFP-15; Le(y)-related carbohydrate antigen; CA 125; urokinase-type plasminogen activator (uPA) and uPA related antigens and complexes (e.g., LMW-uPA, HMW-uPA, uPA aminoterminal fragment (ATF), uPA receptor (UPAR) and complexes with inhibitors such as PA1-1 and PA1-2); beta-glucuronidase; CD31; CD44 splice variants; blood group antigens (e.g., ABH, Lewis, and MN); and genetic lesions or altered expression levels of CCND1, EMS1, BRCA1 and BRCA2 genes.
In summary, the evaluation of proliferation markers, oncogenes, growth factors and growth factor receptors, angiogenic factors, proteases, adhesion factors and tumor suppressor genes, among other cancer markers, can provide important information concerning the risk, presence, status or future behavior of cancer in a patient. Determining the presence or level of expression or activity of one or more of these cancer markers can aid in the differential diagnosis of patients with uncertain clinical abnormalities, for example by distinguishing malignant from benign abnormalities. Furthermore, in patients presenting with established malignancy, cancer markers can be useful to predict the risk of future relapse, or the likelihood of response in a particular patient to a selected therapeutic course. Even more specific information can be obtained by analyzing highly specific cancer markers, or combinations of markers, which may predict responsiveness of a patent to specific drugs or treatment options.
Methods for detecting and measuring cancer markers have been recently revolutionized by the development of immunological assays, particularly by assays that utilize monoclonal antibody technology. Previously, many cancer markers could only be detected or measured using conventional biochemical assay methods, which generally require large test samples and are therefore unsuitable in most clinical applications. In contrast, modern immunoassay techniques can detect and measure cancer markers in relatively much smaller samples, particularly when monoclonal antibodies that specifically recognize a targeted marker protein are used. Accordingly, it is now routine to assay for the presence or absence, level, or activity of selected cancer markers by immunohistochemically staining breast tissue specimens obtained via conventional biopsy methods. Because of the highly sensitive nature of immunohistochemical staining, these methods have also been successfully employed to detect and measure cancer markers in smaller, needle biopsy specimens which require less invasive sample gathering procedures compared to conventional biopsy specimens. In addition, other immunological methods have been developed and are now well known in the art which allow for detection and measurement of cancer markers in non-cellular samples such as serum and other biological fluids from patients. The use of these alternative sample sources substantially reduces the morbidity and costs of assays compared to procedures employing conventional biopsy samples, which allows for application of cancer marker assays in early screening and low risk monitoring programs where invasive biopsy procedures are not indicated.
For the purpose of breast cancer evaluation, the use of conventional or needle biopsy samples for cancer marker assays is often undesirable, because a primary goal of such assays is to detect the cancer before it progresses to a palpable or mammographically detectable tumor stage. Prior to this stage, biopsies are generally contraindicated, making early screening and low risk monitoring procedures employing such samples untenable. Therefore, there is general need in the art to obtain samples for breast cancer marker assays by less invasive means than biopsy, for example by serum withdrawal.
Efforts to utilize serum samples for breast cancer marker assays have met with limited success, largely because the targeted markers are either not detectable in serum, or because telltale changes in the levels or activity of the markers cannot be monitored in serum. In addition, the presence of breast cancer markers in serum probably occurs at the time of micro-metastasis, making serum assays less useful for detecting pre-metastatic disease. In contrast, fluid within the mammary glands themselves is expected to contain much higher and more biologically relevant levels of breast cancer markers than serum, particularly in view of the fact that 80%-90% of all breast cancers occur within the intraductal epithelium of these glands. Fluid within the breast ducts is expected to contain an assemblage and concentration of hormones, growth factors and other potential markers comparable to those secreted by, or acting upon, the surrounding cells of the alveolar-ductal system. Likewise, mammary fluid is expected to contain cells and solid cellular debris or products that can be used in cytological or immunological assays to evaluate intracellular or cell surface markers that may not be detectable in the liquid fraction of mammary fluid.
Previous attempts to develop non-invasive breast cancer marker assays utilizing mammary fluid samples have included studies of mammary fluid obtained from patients presenting with spontaneous nipple discharge. In one of these studies, conducted by Inaji et al., Cancer 60:3008-3013, 1987, levels of the breast cancer marker carcinoembryonic antigen (CEA) were measured using conventional, enzyme linked immunoassay (ELISA) and sandwich-type, monoclonal immunoassay methods. These methods successfully and reproducibly demonstrated that CEA levels in spontaneously discharged mammary fluid provide a sensitive indicator of nonpalpable breast cancer. In a subsequent study, also by Inaji et al., Jpn. J. Clin. Oncol. 19:373-379, 1989, these results were expanded using a more sensitive, dry chemistry, dot-immunobinding assay for CEA determination. This latter study reported that elevated CEA levels occurred in 43% of patients tested with palpable breast tumors, and in 73% of patients tested with nonpalpable breast tumors. CEA levels in the discharged mammary fluid were highly correlated with intratumoral CEA levels, indicating that the level of CEA expression by breast cancer cells is closely reflected in the mammary fluid CEA content. Based on these results, the authors concluded that immunoassays for CEA in spontaneously discharged mammary fluid are useful for screening nonpalpable breast cancer.
Although the evaluation of mammary fluid has been shown to be a useful method for screening nonpalpable breast cancer in women who experience spontaneous nipple discharge, the rarity of this condition renders the methods of Inaji et al, inapplicable to the majority of women who are candidates for early breast cancer screening. In addition, the first Inaji report cited above determined that certain patients suffering spontaneous nipple discharge secrete less than 10 xcexcl of mammary fluid, which is a critically low level for the ELISA and sandwich immunoassays employed in that study. It is likely that other antibodies used to assay other cancer markers may exhibit even lower sensitivity than the anti-CEA antibodies used by Inaji and coworkers, and may therefore not be adaptable or sensitive enough to be employed even in dry chemical immunoassays of small sampbs of spontaneously discharged mammary fluid.
In view of the above, an important need exists in the art for more widely applicable, non-invasive methods and materials to obtain biological samples for use in evaluating, diagnosing and managing breast disease including cancer, particularly for screening early stage, nonpalpable breast tumors. A related need exists for methods and materials that utilize such readily obtained biological samples to evaluate, diagnose and manage breast disease, particularly by detecting or measuring selected breast cancer markers, or panels of breast cancer markers, to provide highly specific, cancer prognostic and/or treatment-related information, and to diagnose and manage pre-cancerous conditions, cancer susceptibility, breast infections and other breast diseases.
It is therefore an object of the present invention to provide non-invasive methods and kits for obtaining biological samples that can be employed in assays for evaluating, diagnosing and managing breast disease, particularly cancer.
It is a further object of the invention to achieve the above object in assay methods and kits that are widely applicable to a broad range of patients, and that include useful assays and kits for screening early stage, nonpalpable mammary tumors.
It is yet another object of the invention to provide methods and kits that utilize the aforementioned biological samples to evaluate, diagnose and manage breast disease, preferably breast cancer, by detecting and/or measuring selected breast disease markers such as breast cancer markers, or panels of breast cancer markers, to provide highly specific prognostic and/or treatment-related information to the clinician.
The invention achieves these objects and other objects and advantages that will become apparent from the description which follows by providing non-invasive methods and devices for obtaining biological samples from a mammary organ of a mammalian patient. Through the use of novel, specialized breast pump devices of the invention, which are fluidly connected with (i.e., by direct or indirect coupling) a solid phase sample collection medium, the physician can rapidly and non-invasively collect mammary fluid samples from lactating or non-lactating female patients without additional intervention. Alternate methods of the invention for mammary fluid sample collection may involve administration of oxytocin, or an oxytocin analog, in an amount effective to stimulate or increase expression of mammary fluid induced in conjunction with employment of the breast pump device. The oxytocin or oxytocin analog (for example a long-acting oxytocin analog such as carbetocin) is administered in a manner (e.g., intranasally) and amount sufficient to reach and stimulate a target alveolar-ductal tissue of the breast, whereby the oxytocin stimulates myoepithelial contraction of the alveolar-ductal tissue to induce or facilitate mammary fluid expression. Alternatively, an intramuscular or intravascular injection of oxytocin can effect the same myoepithelial contraction response as the intranasal administration route. The amount, timing and/or mode of oxytocin administration may be adjusted on an individual basis depending on such factors as menstrual cycle stage, use of birth control or hormone replacement therapy, pregnancy history, age of onset of menarche, ethnicity and other factors known to affect an individual""s propensity for breast fluid expression.
Mammary fluid collection devices of the invention are effective to induce mammary fluid expression for sample collection, alone or in conjunction with oxytocin stimulation. These devices are typically provided as a specialized breast pump which can be applied to the breast covering the nipple, and which typically directly receives the expressed mammary fluid. In preferred methods involving use of a breast pump, negative pressure is generated on the breast to induce expression of mammary fluid, optionally facilitated by prior or concurrent administration of oxytocin. In yet additional alternative methods, mammary fluid can be expressed and collected without the aid of a breast pump, which may require an increase of oxytocin dosage or lengthening of the post administration time period before the mammary fluid is fully expressed from the nipple.
During or after mammary fluid expression, a biological sample is collected from the expressed mammary fluid, which sample may consist of whole mammary fluid, whole cells, cell fragments, cell membranes, selected liquid, cellular or other solid fractions of the mammary fluid, as well as proteins, glycoproteins, peptides, nucleotides (including DNA and RNA polynucleotides) and other like biochemical and molecular constituents of the mammary fluid.
Sample collection can be achieved simply by receiving the expressed mammary fluid within any suitable reservoir, such as an ordinary sample storage container or assay vessel. In preferred embodiments of the invention, the expressed mammary fluid is exposed to a solid phase sample collection medium, simultaneous with or subsequent to the time of breast fluid expression. Suitable solid phase media in this context include microscopic glass slides, capillary tubes, coated tubes, microtiter wells or plates, membranes, filters, affinity columns, dot blot matrices, beads, microspheres, resins, and other like media that will selectively adsorb, bind, filter, partition or otherwise process desired components of the mammary fluid for convenient incorporation into a desired assay. Often it will be desirable to combine a plurality of solid phase media for sample collection, e.g., a filter and membrane, a membrane and a particulate medium, etc., for example to differentially partition and adsorb selected components of the breast fluid.
In conjunction with sample collection, the sample may be exposed to other agents such as buffers, diluents, extraction or chromatographic media, cross-linking agents, denaturing agents, etc., to stabilize or otherwise prepare the sample for processing within a desired assay.
Thus provided within the invention are methods and devices for obtaining a biological sample from a patient and/or determining the amount of a breast disease marker in a biological sample from breast fluid, which employ a novel breast pump or breast pump adapter. The breast pump functions in a similar fashion as a conventional breast pump but also provides a solid phase sample collection medium in fluid connection with the pump. The solid phase sample collection medium may be integrated within the breast pump or otherwise fluidly connected therewith, so that a sample of expressed mammary fluid contacts the collection medium, typically while the pump remains applied to the breast. In more detailed aspects of the invention, methods for employing the novel breast pump include a step of applying the breast pump to stimulate breast fluid expression, with or without prior oxytocin or carbetocin induction, wherein the solid phase sample collection medium is fluidly connected with a breast engaging portion or member of the breast pump.
According to these methods, operation of the pump results in an expressed breast fluid sample contacting the solid phase sample collection medium, typically while the pump remains applied to the breast. Within the foregoing methods, additional methods are provided which employ a novel, hand-held breast pump device, wherein a doctor, technician or patient collecting a breast fluid specimen can grasp and operate the hand-held pump to stimulate expression of breast fluid and collect a specimen thereof while keeping one hand free for additional tasks. The compact hand-held pump design allows the device to be picked up and manipulated with one hand, to seat the breast engaging element against the breast, apply vacuum pressure to the breast by manual operation of the vacuum pump to cause a suitable volume of breast fluid to be expressed at or near the nipple, and to simultaneously collect at least a primary sample of expressed breast fluid onto, or within, the solid phase sample collection medium without additional manual steps or a need to remove the device from the breast or engage two hands in the operation.
In certain collection methods of the invention, breast fluid expressed by use of the general purpose or hand-held breast pump is simultaneously or subsequently diluted, filtered, washed, admixed with fixative or other processing agents, or otherwise processed or modified to yield a collected fluid sample partially or completely devoid of cells, proteins and/or other selected components originally present in the expressed fluid, to provide a processed fluid sample for laboratory analysis. In other embodiments, particulate components of the breast fluid, for example, cells, cellular components and/or cellular debris, are collected after processing and/or modification, e.g., for cytological examination. Often, primary sample collection and/or processing in this context is coincident with the fluid contacting one or more solid phase collection medium(a) fluidly connected with the breast engaging member. Depending on the type(s) of medium(a) used, preliminary sample processing can also be achieved directly by simple operation of the hand-held pump, without the need for additional processing steps or removal of the breast engaging member from the subject""s breast.
In other alternative methods within the invention, preliminary sample processing involves additional steps following breast fluid expression. In certain embodiments, the breast engaging member is removed from the breast after the breast fluid is expressed and the fluid is transferred to a first solid phase sample collection medium, typically a membrane or filter. This initial or primary stage of sample collection may be followed by washing or by manual transfer of selected breast fluid components (e.g., proteins, carbohydrates, cells, or cellular debris) from the first solid phase collection medium (e.g., a nitrocellulose membrane) to a second solid phase medium, e.g., a fluid-containing reservoir. Typically, preliminary sample processing in this regard precedes final packaging of the collected sample for storage or shipment to a lab for further processing and analysis of the sample. In one example, whole cells or other cellular materials are separated from expressed mammary fluid onto a nitrocellulose membrane or a filter, which is typically secured in fluid connection with the breast engaging member by a fixed or removable support member mounted to the engaging member or sample collection housing. The cells are subsequently transferred or washed in fluid (e.g., cytology fluid) to a second solid phase sample collection medium, for example a removable fluid reservoir connected to, or integrated with, the breast engaging member or sample collection housing.
In relation with these methods, various sample collection devices and accessories for use therewith are provided within more detailed embodiments of the invention. Typically, breast fluid collection devices of the invention include a breast engaging member constructed of a non-porous material that is sized and dimensioned to receive at least a nipple portion of a human breast and form a suction seal therewith. One or more solid phase sample collection media are provided in fluid connection with the breast engaging member for receiving a sample of expressed breast fluid. A vacuum pump mechanism is provided in gaseous connection with the breast engaging member for generating negative pressure through the breast engaging member to facilitate breast fluid expression.
In specific embodiments of the collection device of the invention, a sample collection housing is fluidly connected with the breast engaging member. The solid phase sample collection medium is often removably supported within the housing in proximity to the nipple when the breast engaging member is applied to the breast and negative pressure is generated by the vacuum pump mechanism. The solid phase sample collection medium can be one or more microscopic glass slides, capillary tubes, collection tubes, vials, columns, micro-columns, wells, plates, membranes, filters, resins, inorganic matrices, beads, resins, particulate chromatographic media, plastic microparticles, latex particles, coated tubes, coated templates, coated beads, or coated matrices.
Optional features of the breast fluid collection device include removable coupling means for removably coupling said sample collection housing with said breast engaging member. In other embodiments, the solid phase sample collection medium may be supported by a support member integrally or removably mounted within the sample collection housing in fluid connection with said breast engaging member. Various types of support members, including disposable or reuseable discs, cartridges and cassettes are provided as an accessory for use within the invention. In yet additional embodiments, a reciprocating mechanism for reciprocally adjusting a position of the solid phase sample collection medium relative to the breast engaging member is incorporated within the device. The reciprocating mechanism may incorporate a support member or carrier reciprocatingly mounted relative to the breast engaging member, which support member or carrier supports the solid phase sample collection medium. Yet another optional feature of the device includes a breast pump adapter employing concepts of the invention for collection of mammary fluid samples and operable in combination with a conventional breast pump.
In other detailed embodiments of the invention, the sample collection device is a hand-held breast pump incorporating the breast engaging member and the vacuum pump mechanism in a compact, structurally integrated breast fluid collection apparatus suitable for manipulation and operation using only one hand. In certain embodiments, handheld breast pump comprises a modular device made up of a plurality of components, each joined or securable in fixed structural interconnection with one another and capable of partial or complete disassembly from remaining components to facilitate operation, cleaning, servicing and/or storage of the device. The modular breast pump can include, for example, a separate breast engaging member detachable from one or more interconnecting components of the device for cleaning or to allow interchanging of different engaging members to accommodate breast anatomy differences among patients.
Within more detailed embodiments of the hand-held breast pump, the solid phase sample collection medium can be supported by a support member removably mounted in fluid connection with the breast engaging member. The support member can be a removable cassette for removable placement in fluid connection with the breast engaging member. The support member can house any of the above identified collection media, and may incorporate one or more air channels that pass through a body of the support member for passage of vacuum pressure and/or sample materials between the breast engaging member and a sample collection housing member of the hand-held breast pump.
Within other detailed embodiments, the hand-held device includes a fluid-retaining recess, well or reservoir integrated or fluidly connected with the support member or a sample collection housing member of the device. The fluid-retaining recess, well or reservoir may comprise an integral, defined compartment or enclosure within the sample collection housing for receiving the breast fluid and/or constituent samples thereof. Alternatively, fluid-retaining recess, well or reservoir comprises a removable fluid reservoir member of the sample collection housing, typically provided as a rigid sample collection tube or vial removably connected with an outer casing member of the housing. The removable reservoir member is optionally sealably connected with the outer casing member of the housing to form an airtight coupling therewith. In certain embodiments, the removable reservoir member features a circumferential ridge, fin, O-ring or other sealable engagement means to engage and make an airtight seal against a wall or other surface of the casing member when the vial is nested within the casing member.
In additional detailed embodiments, the removable reservoir member is gaseously and fluidly connected with the breast engaging member to facilitate sample collection. For example, the vacuum pressure from the vacuum pump means may be routed to the breast engaging member through the removable reservoir member of the housing, which is modified to include one or more air ports that form a gaseous connection between a lumen of the reservoir and the vacuum pump means. The reservoir member may function in this context as both a conduit for vacuum pressure transmission to the breast and a receptacle for fluid sample materials to directly collect expressed fluid or as a secondary collection medium to receive primarily collected sample materials washed or otherwise transferred from a primary solid phase sample collection medium into the reservoir. For example, a primary solid phase sample collection medium fluidly connected with the breast engaging member may be positioned to collect a primary sample of one or more breast fluid components which can thereafter be washed or otherwise transferred directly or indirectly into the removable reservoir member, without removal or disassembly of the breast engaging member and reservoir member.
The fluid collection reservoir may serve a dual purpose for collection, as well as for storage, transport and/or processing of collected breast fluid or breast fluid component samples. Relating to this purpose, the removable reservoir member further comprises closure means for closing the reservoir after sample collection is completed to prevent sample contamination and spillage. The closure means may comprises a cap adapted to sealably engage a top end of the removable reservoir member. Where the reservoir member is modified to include one or more air ports for transmission of vacuum pressure between the lumen of the reservoir and the vacuum pump means, the closure means include secondary closure means to sealably close the air port(s) after sample collection. For example, the secondary closure means may comprise an adhesive seal or sticker sized and constructed to adhere to an outer wall of the reservoir member surrounding an air port opening. Typically, the secondary closure means comprises a combined closure and labeling device which functions as a secondary closure mechanism to seal the air port(s) of the removable reservoir, and as a labeling template to provide a writing surface for sample labeling. The combined closure and labeling tab or sticker generally includes a first, closure-forming surface for application over the air port to form a seal by juxtaposition or adhesive contact with an outer wall of the removable reservoir, and a second, labeling surface opposite the closure-forming surface made of a blank template material suitable for receiving a stable, ink or graphite imprint. In more detailed aspects, the secondary closure means comprises a combined closure and labeling tab or sticker which is pre-attached to the removable reservoir member in a first, open configuration and which can be manually repositioned or otherwise manipulated after sample collection to a second, closed configuration to form a seal or closure against the air port(s).
In related aspects of the invention, a novel breast fluid collection reservoir, e.g., a modified cytology vial, is provided for use within a mammary fluid collection device of the invention, which reservoir incorporates the foregoing features of the removable reservoir member of the sample collection housing. The novel collection reservoir thus provided is useful within the breast fluid collection methods of the invention, as well as within sample processing and diagnostic assay methods performed in the laboratory subsequent to collection of a breast fluid sample.
In related aspects of the invention, methods are provided for determining the presence or amount of a breast disease marker, preferably a breast cancer marker, in biological samples obtained from a mammary organ of a mammalian patient. These methods may involve intranasal, intramuscular or intravascular administration of oxytocin or an oxytocin analog to mammalian patients in amounts effective to stimulate mammary fluid expression in the patient. Once a sufficient post-administration time period has elapsed to allow the oxytocin to reach and stimulate target alveolar-ductal tissues, mammary fluid is collected directly from the nipple or, alternatively, the breast is pumped, and a biological sample from expressed mammary fluid is collected, as above. After the sample is collected a bioassay is conducted on the sample to determine the presence and/or amount of the breast disease marker in the sample. Suitable bioassays in this regard include assays to detect known markers of breast disease, such as assays employing immunological or other suitable probes to detect specific antigens and other markers expressed by selected pathogens, including bacterial and viral pathogens. More preferred bioassays will detect individual markers or panels of markers of benign breast tumors, pre-cancerous breast disease, and/or breast cancer, such as assays employing immunological or other suitable probes to detect specific antigens and other markers expressed by benign, pre-cancerous and/or cancerous alveolar-ductal cells of the breast. Preferably, the assay will detect the presence or amount of multiple breast disease markers in the biological sample, for example by including a panel of immunological or molecular probe(s) that bind or react with multiple breast cancer markers.
Within further related aspects of the invention, novel methods are provided handling or processing a biological sample of mammary fluid, or a component thereof, for use in a diagnostic assay to detect or quantify a breast disease marker in a biological sample of mammary fluid. The methods typically include providing or obtaining the biological sample of mammary fluid, or component thereof, in a fluid-retaining reservoir that features a top end defining a primary opening for access to the sample and an outer reservoir wall defining one or more air ports that communicate between the outer wall and an inner lumen of the vial. In certain aspects, the fluid-containing reservoir member includes closure means for closure of the reservoir after the sample is introduced therein, to prevent sample contamination and spillage.
The closure means may comprise a cap adapted to sealably engage a top end of the removable reservoir member. In various embodiments, the closure includes secondary closure means to sealably close the air port(s) after the biological sample is introduced into said reservoir. Exemplary secondary closure means include an adhesive seal or sticker that is sized and constructed to adhere to an outer wall of the reservoir member surrounding an air port opening. Alternatively, the secondary closure means may include a combined closure and labeling device which functions as a secondary closure mechanism to seal the air port(s) of the removable reservoir and as a labeling template to provide a writing surface for sample labeling. The combined closure and labeling tab or sticker may be directly applied to seal the air port after sample collection having a first, closure-forming surface for application over the air port to form a seal by juxtaposition or adhesive contact with an outer wall of the removable reservoir, and a second, labeling surface opposite the closure-forming surface made of a blank template material suitable for receiving a stable, ink or graphite imprint thereon.
Within additional aspects of the invention, the first, closure-forming surface of the reservoir can be provided with an adhesive coating resistant to disruption by contact with aqueous solutions. The secondary closure means may include a combined closure and labeling tab or sticker which is pre-attached to the removable reservoir member in a first, open configuration and which can be manually repositioned or otherwise manipulated after sample collection to a second, closed configuration to form a seal or closure against the air port(s). The secondary closure means can optionally comprise an adhesive tab or strip folded in the open configuration to form an inner layer affixed to the reservoir proximate to the air port, and an outer layer folded over the inner layer, said outer layer providing the first, closure-forming surface and the second, labeling surface, wherein the outer layer can be unfolded away from the inner layer and wrapped around the reservoir so that the closure-forming surface covers the air port to form a fluid-resistant closure and the labeling surface faces outward for recordation of sample data. The outer layer can be optionally secured in a folded-back position against the inner layer by adhesive engagement of the labeling surface with the inner layer.
In related embodiments, the first, closure-forming surface may be provided with an adhesive coating that is protected in the open configuration by folding of an end segment of the outer layer bearing the adhesive coating back, so that the closure forming surface provides a protective surface to shield the adhesive prior to closure, whereby the end segment can be lifted and pulled outward to unfold the end segment to separate the adhesive coating on the closure-forming surface from the protective surface and to release the outer layer from the inner layer for closing of the air port(s).
In certain aspects of the invention, the foregoing methods utilize a fluid-retaining reservoir that is a modified cytology vial. In other aspects, the fluid-retaining reservoir comprises a removable fluid reservoir member of a sample collection housing of a mammary fluid collection device. The fluid-retaining reservoir may be a rigid sample collection tube or vial that is optionally removably connected with an outer casing member of a sample collection housing of said collection device. The fluid-retaining reservoir can alternately be adapted for removable, sealable connection with the outer casing member of the housing to form an airtight coupling therewith. In exemplary embodiments, the fluid-retaining reservoir is a cytology vial that is sealably connectable with said outer casing member to form an airtight coupling therewith. The fluid-retaining reservoir can optionally be removably nested within the casing member to form a substantially airtight contact between an inner wall of the casing member and an outer wall, or a top or bottom end, of the reservoir member. In certain configurations, an outer wall of the fluid-retaining reservoir bears a circumferential ridge, fin or O-ring. The fluid-retaining reservoir can optionally comprise a removable fluid reservoir member of the sample collection housing of a mammary fluid collection device, and the above-described circumferential ridge, fin or O-ring may be adapted to engage and make a circumferential airtight seal against an inner wall of a casing member of the sample collection housing of the device.
In exemplary sample handling and processing methods of the invention, the mammary fluid sample or component thereof includes whole cells or cell fragments. The handling and processing methods can further include the step of accessing the sample within reservoir to transfer or process the sample for detection or quantification of the breast disease marker. In exemplary embodiments, the sample may be transferred to a second reservoir or other sample container or template for processing the sample to detect or quantify the breast disease marker. Exemplary steps of sample processing include detection or quantification of the breast disease marker by staining cells or cell fragments from the sample with a cytological stain to detect a cytological marker. Alternative processing steps include detection or quantification of the breast disease marker by microscopic examination of stained cells or cell fragments from the sample. Within certain embodiments, the biological sample includes one or more components selected from the group consisting of whole mammary fluid, whole cells, cell fragments, cell membranes, purified proteins, bulk proteins, glycoproteins, peptides and polynucleotide components of a primary mammary fluid sample. Often, the breast disease marker will comprise a breast cancer marker. In various aspects, the breast disease marker is selected from the group consisting of a protein, a peptide, a glycoprotein, a lipid, a DNA polynucleotide and an RNA polynucleotide. Exemplary breast disease markers include Ki67 Growth Factor, Cyclin D1, Proliferating Cell Nuclear Antigen, Transforming Growth Factor, Tissue Plasminogen Activator, Insulin Growth Factor Receptors, Collagenase Type IV, Laminin Receptor, Integrins, p53, rb, nm23, ras, c-myc, Heat Shock Proteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin, CEA, HMFG, MCA, vasopressin and cathepsin.
Within the foregoing methods the breast disease markers may be detected using any of a variety of useful techniques, for example by ELISA immunoassay, immunoprecipitation assay, or solid phase immunoassay.
In yet additional aspects of the invention, clinically useful kits are provided for determining the presence and/or amount of a breast disease marker, preferably a breast cancer marker, in biological samples obtained from a mammary organ of a mammalian patient. The kits include a mammary fluid collection device in the form of a general purpose or hand-held breast pump as described herein. Additional kits include one or more breast pump attachments (e.g., a detachable breast engaging member, or multiple such attachments for use with different patients), accessories (e.g., replaceable fluid-retaining reservoirs), solid phase media, and/or disposable or reusable support members, cartridges or cassettes for holding collection media, as described herein. These and other kit components may be provided, alone or in any combination, with or without inclusion of the basic breast pump apparatus in the kit. Yet additional kits include a pharmaceutical preparation of oxytocin or an oxytocin analog in a biologically suitable carrier for use in alternate mammary fluid collection methods of the invention. Still other kits include on or more preparative and/or diagnostic reagents selected from those disclosed herein, including one or more fixatives, probes, labels and the like in separate or common containers. In certain embodiments of the invention, kits include compositions and/or devices for detecting the presence or amount of one or more breast disease marker(s) in the biological sample, often including one or more immunological or molecular probe(s) that binds or reacts with one or more breast cancer marker(s). The foregoing kit components are generally assembled in a collective packaging unit, which may include written or otherwise user-accessible instructions detailing the sample collection, handling and/or processing methods of the invention.